Polynucleotide sequences and proteins encoded thereby useful for modifying plant characteristics

ABSTRACT

The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides. The present invention further relates to the uses of these nucleic acid molecules and polypeptides. For example, the nucleic acid molecules and polypeptides could be used in making enzymes or used to make plants, plant cells, plant materials or seeds of a plant having such modulated growth or phenotype characteristics that are altered with respect to wild type plants grown under similar conditions.

CROSS REFERENCE PARAGRAPH

This application is a Continuation of copending application Ser. No.13/626,508 filed on Sep. 25, 2012, which is a Divisional of copendingapplication Ser. No. 12/168,788 (now U.S. Pat. No. 8,299,318) filed onJul. 7, 2008, and for which priority is claimed under 35 U.S.C. § 120;and this application claims priority of U.S. Provisional Application No.60/958,715 filed on Jul. 5, 2007 under 35 U.S.C. § 119(e); the entirecontents of each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to isolated nucleic acid molecules andtheir corresponding encoded polypeptides. The present invention furtherrelates to using the nucleic acid molecules and polypeptides to maketransgenic plants, plant cells, plant materials or seeds of a planthaving modulated phenotypic and growth characteristics as compared towild-type plants grown under similar conditions.

BACKGROUND OF THE INVENTION

Switchgrass is a hardy, warm season perennial bunch grass of the milletfamily. It was a component of the native prairie in the Great Plainsstates and Canada and can grow up to 1.8-22 m in height. Switchgrasspropagates by rhizomes and seeds produced on spikelets. Switchgrassgrown from seed has low vigor and is slow to establish, thus productionis decreased until the third year growing year. Switchgrass uses the C4carbon fixation pathway which allows for improved water use efficiencyduring its growth period, providing an advantage under drought and hightemperature conditions. Once established, switchgrass is also tolerantof flooding and grows rapidly, capturing a significant amount of solarenergy and turning it into stored energy in the form of cellulose.

As pasture forage, switchgrass is palatable in the vegetative stage andis grazed by certain animals, used as ground cover to control erosionand farmed as forage for livestock.

Switchgrass is also a good energy crop and can be cut and baled withconventional mowers and balers. Once established in a field, it can beharvested as a cash crop, either annually or semiannually, for 10 yearsor more before replanting is needed. Switchgrass offers importantadvantages as an energy crop, in part because it can be liquified,gasified, or burned directly. Ethanol production from switchgrass canprovide as much as twenty times more net energy output than corn andremoves considerably more CO₂ from the air. Switchgrass has thepotential to produce the biomass required for production of up to 100gallons (380 liters) of ethanol per metric ton, which gives switchgrassthe potential to produce 1000 gallons of ethanol per acre, compared to665 gallons for the sucrose from sugarcane and 400 gallons for thestarch from corn.

Combustion of switchgrass pellets can result in nearly completecombustion with only 3% to 4% of original mass remaining as ash due inpart to switchgrass' lower silica and chloride content as compared tocool season grasses. Ash contents can be further reduced by allowingswitchgrass to overwinter in the field, thereby reducing the silica andchloride contents further through the process of leaching. There arealso advantages from an ash content perspective to producing switchgrassin sandy soils as opposed to clay soils, again based on silica andchloride contents.

Despite the many advantages that switchgrass has as an energy crop, inorder for this grass to fulfill its promise, new varieties ofswitchgrass are needed that will have increased hardiness and yield,reduce the need for nitrogen and other chemical fertilizers, and allowpropagation under widely variant growing conditions.

Plants specifically improved for energy usage can be obtained usingmolecular technologies. Manipulation of crop performance has beenaccomplished conventionally for centuries through plant breeding. Thebreeding process is, however, both time-consuming and labor-intensive.Furthermore, appropriate breeding programs must be specially designedfor each relevant plant species.

On the other hand, molecular genetics approaches that introduce andexpress recombinant nucleic acid molecules allow production of plantspecies tailored to grow more efficiently and produce more product inunique geographic and/or climatic environments. To this end, there is anongoing need for genetic sequences and materials to advantageouslymanipulate plant characteristics such as architecture, biomass,development, composition, conversion efficiency, energy output,confinement, nitrogen use, nutrient uptake, phosphate use,photosynthetic capacity, shade avoidance, cold tolerance, droughttolerance, water use efficiency, stress tolerance, vigor, flowering timeand yield to maximize the benefits of energy crops and othereconomically important crops depending on the benefit sought and theparticular environment in which the crop must grow. These molecules maybe from the plant itself, and simply expressed at a higher or lowerlevel, or the molecules may be from different plant species.

SUMMARY OF THE INVENTION

The present invention, therefore, relates to isolated nucleic acidmolecules and polypeptides and their use in making transgenic plants,plant cells, plant materials or seeds of plants having modulated plantcharacteristics, with respect to wild-type plants grown under similar oridentical conditions, in traits such size (in whole or in part),vegetative growth, increased organ number and/or increased biomass(sometimes hereinafter collectively referred to as increased biomass),lethality, sterility, stress tolerance, or compositional characteristicsas compared to a wild-type plant cultivated under identical conditions.(sometimes hereinafter collectively referred to as “modulated growth andphenotype characteristics”).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

DETAILED DESCRIPTION OF THE INVENTION 1. The Invention

The invention of the present application may be described by, but notnecessarily limited to, the following exemplary embodiments.

The present invention discloses novel isolated nucleic acid molecules,nucleic acid molecules that interfere with these nucleic acid molecules,nucleic acid molecules that hybridize to these nucleic acid molecules,and isolated nucleic acid molecules that encode the same protein due tothe degeneracy of the DNA code. Additional embodiments of the presentapplication further include the polypeptides encoded by the isolatednucleic acid molecules of the present invention.

More particularly, the nucleic acid molecules of the present inventioncomprise: (a) a nucleotide sequence encoding an amino acid sequence thatis at least 85% identical to any one of the polypeptides in the sequencelisting (SEQ ID Nos. 1-9762), (b) a nucleotide sequence encoding anamino acid sequence that is at least 85% identical to any one of thepolypeptides from Panicum virgatum in the sequence listing (SEQ ID Nos.1-9176), (c) a nucleotide sequence that is complementary to any one ofthe nucleotide sequences according to (a) and (b), (d) a nucleotidesequence according to any one of the nucleotides in the sequence listingSEQ ID Nos. 1-9176, (e) a nucleotide sequence able to interfere with anyone of the nucleotide sequences according to (a) and (b), (f) anucleotide sequence able to form a hybridized nucleic acid duplex withthe nucleic acid according to any one of paragraphs (a)-(d) at atemperature from about 40° C. to about 48° C. below a meltingtemperature of the hybridized nucleic acid duplex, (g) a nucleotidesequence encoding any one of the polypeptide sequences from Panicumvirgatum in the sequence listing, (h) a nucleotide sequence encoding anyone of the polypeptide sequences given in the sequence listing.

The present invention further embodies a vector comprising a firstnucleic acid having a nucleotide sequence encoding a plant transcriptionand/or translation signal, and a second nucleic acid having a nucleotidesequence according to the isolated nucleic acid molecules of the presentinvention. More particularly, the first and second nucleic acids may beoperably linked. Even more particularly, the second nucleic acid may beendogenous to a first organism, and any other nucleic acid in the vectormay be endogenous to a second organism. Most particularly, the first andsecond organisms may be different species.

In a further embodiment of the present invention, a host cell maycomprise an isolated nucleic acid molecule according to the presentinvention. More particularly, the isolated nucleic acid molecule of thepresent invention found in the host cell of the present invention may beendogenous to a first organism and may be flanked by nucleotidesequences endogenous to a second organism. Further, the first and secondorganisms may be different species. Even more particularly, the hostcell of the present invention may comprise a vector according to thepresent invention, which itself comprises nucleic acid moleculesaccording to those of the present invention.

In another embodiment of the present invention, the isolatedpolypeptides of the present invention may additionally comprise aminoacid sequences that are at least 85% identical to any one of thepolypeptides in the sequence listing.

Other embodiments of the present invention include methods ofintroducing an isolated nucleic acid of the present invention into ahost cell. More particularly, an isolated nucleic acid molecule of thepresent invention may be contacted to a host cell under conditionsallowing transport of the isolated nucleic acid into the host cell. Evenmore particularly, a vector as described in a previous embodiment of thepresent invention, may be introduced into a host cell by the samemethod.

Methods of detection are also available as embodiments of the presentinvention. Particularly, methods for detecting a nucleic acid moleculeaccording to the present invention in a sample. More particularly, theisolated nucleic acid molecule according to the present invention may becontacted with a sample under conditions that permit a comparison of thenucleotide sequence of the isolated nucleic acid molecule with anucleotide sequence of nucleic acid in the sample. The results of suchan analysis may then be considered to determine whether the isolatednucleic acid molecule of the present invention is detectable andtherefore present within the sample.

A further embodiment of the present invention comprises a plant, plantcell, plant material or seeds of plants comprising an isolated nucleicacid molecule and/or vector of the present invention. More particularly,the isolated nucleic acid molecule of the present invention may beexogenous to the plant, plant cell, plant material or seed of a plant.

A further embodiment of the present invention includes a plantregenerated from a plant cell or seed according to the presentinvention. More particularly, the plant, or plants derived from theplant, plant cell, plant material or seeds of a plant of the presentinvention preferably has increased size (in whole or in part), increasedvegetative growth, increased organ number and/or increased biomass(sometimes hereinafter collectively referred to as increased biomass),lethality, sterility, improved stress tolerance, or compositionalcharacteristics as compared to a wild-type plant cultivated underidentical conditions. Furthermore, the transgenic plant may comprise afirst isolated nucleic acid molecule of the present invention, whichencodes a protein involved in modulating growth and phenotypecharacteristics, and a second isolated nucleic acid molecule whichencodes a promoter capable of driving expression in plants, wherein thegrowth and phenotype modulating component and the promoter are operablylinked. More preferably, the first isolated nucleic acid may bemis-expressed in the transgenic plant of the present invention, and thetransgenic plant exhibits modulated characteristics as compared to aprogenitor plant devoid of the nucleic acid, when the transgenic plantand the progenitor plant are cultivated under identical environmentalconditions. In another embodiment of the present invention the modulatedgrowth and phenotype characteristics may be due to the inactivation of aparticular sequence, using for example an interfering RNA.

A further embodiment consists of a plant, plant cell, plant material orseed of a plant according to the present invention which comprises anisolated nucleic acid molecule of the present invention, wherein theplant, or plants derived from the plant, plant cell, plant material orseed of a plant, has the modulated growth and phenotype characteristicsas compared to a wild-type plant cultivated under identical conditions.

Another embodiment of the present invention includes methods ofmodulating growth and phenotype characteristics in plants. Moreparticularly, these methods comprise transforming a plant with anisolated nucleic acid molecule according to the present invention.

In yet another embodiment, lethality genes of the invention can be usedto control transmission and expression of transgenic traits, therebyfacilitating the cultivation of transgenic plants without the undesiredtransmission of transgenic traits to other plants. Such lethality genescan also be utilized for selective lethality, by combining the lethalgene with appropriate promoter elements for selective expression, tothereby cause lethality of only certain cells or only under certainconditions.

In another aspect, methods of identifying a trait associatedpolymorphism are provided. The methods include providing a population ofswitchgrass plants, and determining whether one or more polymorphisms inthe population are present within a nucleic acid corresponding to aswitchgrass polynucleotide provided in the Sequence Listing. Thecorrelation between variation in the trait in a tissue in plants of thepopulation and the presence of the one or more polymorphisms in plantsof the population is measured, thereby permitting identification of thetrait associated polymorphism. The trait may be selected from a featurenoted in the Sequence Listing for a polypeptide encoded by thecorresponding switchgrass polynucleotide.

2. Definitions

The following terms are utilized throughout this application:

Biomass: As used herein, “biomass” refers to useful biological materialincluding a product of interest, which material is to be collected andis intended for further processing to isolate or concentrate the productof interest. “Biomass” may comprise the fruit, or parts of it, or seeds,leaves, or stems or roots where these are the parts of the plant thatare of particular interest for the industrial purpose. “Biomass”, as itrefers to plant material, includes any structure or structures of aplant that contain or represent the product of interest.

Transformation: Examples of means by which this can be accomplished aredescribed below and include Agrobacterium-mediated transformation (ofdicots (9-10), of monocots (11-13), and biolistic methods (14)),electroporation, in planta techniques, and the like. Such a plantcontaining the exogenous nucleic acid is referred to here as a T₀ forthe primary transgenic plant and T₁ for the first generation.

Functionally Comparable Proteins or Functional Homologs: This phrasedescribes a set of proteins that perform similar functions within anorganism. By definition, perturbation of an individual protein withinthat set (through misexpression or mutation, for example) is expected toconfer a similar phenotype as compared to perturbation of any otherindividual protein. Such proteins typically share sequence similarityresulting in similar biochemical activity. Within this definition,homologs, orthologs and paralogs are considered to be functionallycomparable.

Functionally comparable proteins will give rise to the samecharacteristic to a similar, but not necessarily the same, degree.Typically, comparable proteins give the same characteristics where thequantitative measurement due to one of the comparables is at least 20%of the other, more typically, between 30 to 40%; even more typically,between 50-60%; even more typically between 70 to 80%; even moretypically between 90 to 100% of the other.

Heterologous sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequence, areconsidered heterologous to said coding sequence. Elements operativelylinked in nature and contiguous to each other are not heterologous toeach other. On the other hand, these same elements remain operativelylinked but become heterologous if another filler sequence is placedbetween them. Thus, the promoter and coding sequences of a corn geneexpressing an amino acid transporter are not heterologous to each other,but the promoter and coding sequence of a corn gene operatively linkedin a novel manner are heterologous.

Misexpression: The term “misexpression” refers to an increase or adecrease in the transcription of a coding region into a complementaryRNA sequence as compared to the wild-type. This term also encompassesexpression and/or translation of a gene or coding region or inhibitionof such transcription and/or translation for a different time period ascompared to the wild-type and/or from a non-natural location within theplant genome, including a gene coding region from a different plantspecies or from a non-plant organism.

Percentage of sequence identity: As used herein, the term “percentsequence identity” refers to the degree of identity between any givenquery sequence and a subject sequence. A query nucleic acid or aminoacid sequence is aligned to one or more subject nucleic acid or aminoacid sequences using the computer program ClustalW (version 1.83,default parameters), which allows alignments of nucleic acid or proteinsequences to be carried out across their entire length (globalalignment). Chenna et al. (2003) Nucleic Acids Res. 31(13):3497-500.

ClustalW calculates the best match between a query and one or moresubject sequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For an alignment of multiple nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launcherwebsite and at the European Bioinformatics Institute website on theWorld Wide Web.

To determine a percent identity for polypeptide or nucleic acidsequences between a query and a subject sequence, the sequences arealigned using Clustal W and the number of identical matches in thealignment is divided by the query length, and the result is multipliedby 100. The output is the percent identity of the subject sequence withrespect to the query sequence. It is noted that the percent identityvalue can be rounded to the nearest tenth. For example, 78.11, 78.12,78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17,78.18, and 78.19 are rounded up to 78.2.

In some embodiments, the amino acid sequence of a suitable subjectpolypeptide has greater than 40% sequence identity(e.g., >40%, >50%, >60%, >70% or >80%, >90%,91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) to the aminoacid sequence of the query polypeptide. In other embodiments, thenucleotide sequence of a suitable subject nucleic acid has greater than40% sequence identity(e.g., >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%,or >99%) to the nucleotide sequence of the query nucleic acid.

Regulatory Regions: The term “regulatory region” refers to nucleotidesequences that, when operably linked to a sequence, influencetranscription initiation or translation initiation or transcriptiontermination of said sequence and the rate of said processes, and/orstability and/or mobility of a transcription or translation product. Asused herein, the term “operably linked” refers to positioning of aregulatory region and said sequence to enable said influence. Regulatoryregions include, without limitation, promoter sequences, enhancersequences, response elements, protein recognition sites, inducibleelements, protein binding sequences, 5′ and 3′ untranslated regions(UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, and introns. Regulatory regions can beclassified in two categories, promoters and other regulatory regions.

Stringency: “Stringency,” as used herein is a function of nucleic acidmolecule probe length, nucleic acid molecule probe composition (G+Ccontent), salt concentration, organic solvent concentration andtemperature of hybridization and/or wash conditions. Stringency istypically measured by the parameter T_(m), which is the temperature atwhich 50% of the complementary nucleic acid molecules in thehybridization assay are hybridized, in terms of a temperaturedifferential from T_(m). High stringency conditions are those providinga condition of T_(m)-5° C. to T_(m)-10° C. Medium or moderate stringencyconditions are those providing T_(m)-20° C. to T_(m)-29° C. Lowstringency conditions are those providing a condition of T_(m)-40° C. toT_(m)-48° C. The relationship between hybridization conditions and T_(m)(in ° C.) is expressed in the mathematical equation:

T _(m)=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)  (I)

where N is the number of nucleotides of the nucleic acid molecule probe.This equation works well for probes 14 to 70 nucleotides in length thatare identical to the target sequence. The equation below, for T_(m) ofDNA-DNA hybrids, is useful for probes having lengths in the range of 50to greater than 500 nucleotides, and for conditions that include anorganic solvent (formamide):

T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L0.63(%formamide)  (II)

where L represents the number of nucleotides in the probe in the hybrid(Bonner et al. (1973) J. Mol. Biol. 81:123). The T_(m) of Equation II isaffected by the nature of the hybrid: for DNA-RNA hybrids, T_(m) is10-15° C. higher than calculated; for RNA-RNA hybrids, T_(m) is 20-25°C. higher. Because the T_(m) decreases about 1° C. for each 1% decreasein homology when a long probe is used (Frischauf et al. (1983) J. MolBiol, 170: 827-842), stringency conditions can be adjusted to favordetection of identical genes or related family members.

Equation II is derived assuming the reaction is at equilibrium.Therefore, hybridizations according to the present invention are mostpreferably performed under conditions of probe excess and allowingsufficient time to achieve equilibrium. The time required to reachequilibrium can be shortened by using a hybridization buffer thatincludes a hybridization accelerator such as dextran sulfate or anotherhigh volume polymer.

Stringency can be controlled during the hybridization reaction, or afterhybridization has occurred, by altering the salt and temperatureconditions of the wash solutions. The formulas shown above are equallyvalid when used to compute the stringency of a wash solution. Preferredwash solution stringencies lie within the ranges stated above; highstringency is 5-8° C. below T_(m) medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

T₀: The term “T₀” refers to the whole plant, explant or callus tissue,inoculated with the transformation medium.

T₁: The term T₁ refers to either the progeny of the T₀ plant, in thecase of whole-plant transformation, or the regenerated seedling in thecase of explant or callous tissue transformation.

T₂: The term T₂ refers to the progeny of the T₁ plant. T₂ progeny arethe result of self-fertilization or cross-pollination of a T₁ plant.

T₃: The term T₃ refers to second generation progeny of the plant that isthe direct result of a transformation experiment. T₃ progeny are theresult of self-fertilization or cross-pollination of a T₂ plant.

3. Important Characteristics of the Polynucleotides and Polypeptides ofthe Invention

The nucleic acid molecules and polypeptides of the present invention areof interest because when the nucleic acid molecules are mis-expressed(i.e., when expressed at a non-natural location or in an increased ordecreased amount relative to wild-type) they produce plants that exhibitmodulated growth and phenotype characteristics as compared to wild-typeplants. This trait can be used to exploit or maximize plant products.For example, the nucleic acid molecules and polypeptides of the presentinvention are used to increase the expression of genes that cause theplant to have modulated growth and phenotype characteristics.

Because some of the disclosed sequences and methods increase vegetativegrowth, the disclosed methods can be used to enhance biomass production.For example, plants that grow vegetatively have an increase biomassproduction, compared to a plant of the same species that is notgenetically modified for substantial vegetative growth. Examples ofincreases in biomass production include increases of at least 5%, atleast 10%, at least 20%, or even at least 50%, when compared to anamount of biomass production by a plant of the same species notgenetically modified.

The life cycle of flowering plants in general can be divided into threegrowth phases: vegetative, inflorescence, and floral (late inflorescencephase). In the vegetative phase, the shoot apical meristem (SAM)generates leaves that later will ensure the resources necessary toproduce fertile offspring. Upon receiving the appropriate environmentaland developmental signals the plant switches to floral, or reproductive,growth and the SAM enters the inflorescence phase (I) and gives rise toan inflorescence with flower primordia. During this phase the fate ofthe SAM and the secondary shoots that arise in the axils of the leavesis determined by a set of meristem identity genes, some of which preventand some of which promote the development of floral meristems. Onceestablished, the plant enters the late inflorescence phase (12) wherethe floral organs are produced. If the appropriate environmental anddevelopmental signals for floral, or reproductive, growth are disrupted,the plant will not be able to enter reproductive growth, thereforemaintaining vegetative growth and increasing overall biomass.

As more and more transgenic plants are developed and introduced into theenvironment, it can be important to control the undesired spread of thetransgenic triat(s) from transgenic plants to other traditional andtransgenic cultivars, plant species and breeding lines, therebypreventing cross-contamination. Such genetic confinement systems (alsocalled biocontainment applications) can be designed using a number ofdifferent technical strategies. The use of a conditionally lethal gene,i.e. one which results in plant cell death under certain conditions, hasbeen suggested as a means to selectively kill plant cells containing arecombinant DNA (see e.g., WO 94/03619 and US patent publication20050044596A). The use of genes to control transmission and expressionof transgenic traits is also described in (see US patent publication20050257293A1), which is hereby incorporated by reference. Some of thenucleotides of the invention are lethal genes, and can therefore be usedas conditionally lethal genes, namely genes to be expressed in responseto specific conditions, or in specific plant cells. For example, a genethat encodes a lethal trait can be placed under that control of a tissuespecific promoter, or under the control of a promoter that is induced inresponse to specific conditions, for example, a specific chemicaltrigger, or specific environmental conditions.

Male or female sterile genes can also be used to control the spread ofcertain germplasm, such as by selective destruction of tissue, such asof the tapetum by fusing such a gene to a tapetum-specific promoter suchas, TA29. Further examples of such promoters are described below.

The sequences of the invention can be used to advantageously manipulateplant characteristics in traits such as size (in whole or in part),vegetative growth, increased organ number and/or increased biomass(sometimes hereinafter collectively referred to as increased biomass),lethality, sterility, stress tolerance, or compositional characteristicsas compared to a wild-type plant cultivated under identical conditions.Furthermore, the sequences of the invention can be used toadvantageously manipulate plant characteristics such as architecture,biomass, development, composition, conversion efficiency, energy output,confinement, nitrogen use, nutrient uptake, phosphate use,photosynthetic capacity, shade avoidance, cold tolerance, droughttolerance, water use efficiency, stress tolerance, vigor, flowering timeand yield to maximize the benefits of energy crops and othereconomically important crops depending on the benefit sought and theparticular environment in which the crop must grow. These molecules maybe from the plant itself, and simply expressed at a higher or lowerlevel, or the molecules may be from different plant species

The sequences of the invention can be applied to substrates for use inarray applications such as, but not limited to, assays of global geneexpression, under varying conditions of development, and growthconditions. The arrays are also used in diagnostic or forensic methods.

The polynucleotides of the invention are also used to create varioustypes of genetic and physical maps of the genome of switchgrass plants.Some are absolutely associated with particular phenotypic traits,allowing construction of gross genetic maps. Creation of such maps isbased on differences or variants, generally referred to aspolymorphisms, between different parents used in crosses. Common methodsof detecting polymorphisms that can be used are restriction fragmentlength polymorphisms (RFLPs, single nucleotide polymorphisms (SNPs) orsimple sequence repeats [(SSRs), also called microsatellites].

The sequence information disclosed herein can be useful in breeding ofswitchgrass plants. Based on the information in the Sequence Listing,one can search for and identify polymorphisms linked to genetic loci forsuch polypeptides. As those of skill in the art appreciate,polymorphisms can be identified based on characterization of libraries,such as genomic or expression libraries, and/or characterization ofnucleic acids extracted from individual plants, and possibly amplifiedand/or otherwise processed. For example, the nucleotide sequencesprovided in the sequence listing can serve for design of primers foramplification of nucleic acids and polymorphism characterization. Assuch, polymorphisms may be found in coding regions or untranslatedregions of polynucleotides presented in the Sequence Listing, or theymay be found within the locus for a disclosed sequence. Polymorphismsthat can be identified include simple sequence repeats (SSRs), rapidamplification of polymorphic DNA (RAPDs), amplified fragment lengthpolymorphisms (AFLPs) and restriction fragment length polymorphisms(RFLPs), as described below. If a polymorphism is identified, itspresence and frequency in populations is analyzed to determine if it isstatistically significantly correlated to a trait, such as the traitnoted in the Sequence Listing. Those polymorphisms that are correlatedwith a trait can be incorporated into a marker assisted breeding programto facilitate the development of lines that have a desired alterationthe respective trait. Typically, a polymorphism identified in such amanner is used with polymorphisms at other loci that are correlated withthe same trait.

The use of RFLPs and of recombinant inbred lines for such geneticmapping is described for Arabidopsis by Alonso-Blanco et al. (Methods inMolecular Biology, vol. 82, “Arabidopsis Protocols”, pp. 137-146, J. M.Martinez-Zapater and J. Salinas, eds., c. 1998 by Humana Press, Totowa,N.J.) and for corn by Burr (“Mapping Genes with Recombinant Inbreds”,pp. 249-254. In Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c.1994 by Springer-Verlag New York, Inc.: New York, N.Y., USA; BerlinGermany; Burr et al. Genetics (1998) 118: 519; Gardiner, J. et al.,(1993) Genetics 134: 917). This procedure, however, is not limited toplants and is used for other organisms (such as yeast) or for individualcells.

The polynucleotides of the present invention are also used for simplesequence repeat (SSR) mapping. Rice SSR mapping is described by Morganteet al. (The Plant Journal (1993) 3: 165), Panaud et al. (Genome (1995)38: 1170); Senior et al. (Crop Science (1996) 36: 1676), Taramino et al.(Genome (1996) 39: 277) and Ahn et al. (Molecular and General Genetics(1993) 241: 483-90). SSR mapping is achieved using various methods. Inone instance, polymorphisms are identified when sequence specific probescontained within a polynucleotide flanking an SSR are made and used inpolymerase chain reaction (PCR) assays with template DNA from two ormore individuals of interest. Here, a change in the number of tandemrepeats between the SSR-flanking sequences produces differently sizedfragments (U.S. Pat. No. 5,766,847). Alternatively, polymorphisms areidentified by using the PCR fragment produced from the SSR-flankingsequence specific primer reaction as a probe against Southern blotsrepresenting different individuals (U. H. Refseth et al., (1997)Electrophoresis 18: 1519).

The polynucleotides of the invention can further be used to identifycertain genes or genetic traits using, for example, known AFLPtechnologies, such as in EP0534858 and U.S. Pat. No. 5,878,215.

The polynucleotides of the present invention are also used for singlenucleotide polymorphism (SNP) mapping.

The polynucleotides of the invention can be used with the various typesof maps discussed above to identify Quantitative Trait Loci (QTLs). Manyimportant crop traits, such as the solids content of tomatoes, arequantitative traits and result from the combined interactions of severalgenes. These genes reside at different loci in the genome, often timeson different chromosomes, and generally exhibit multiple alleles at eachlocus. The polynucleotides of the invention are used to identify QTLsand isolate specific alleles as described by de Vicente and Tanksley(Genetics (1993) 4:585). Once a desired allele combination isidentified, crop improvement is accomplished either throughbiotechnological means or by directed conventional breeding programs(for review see Tanksley and McCouch (1997) Science 277:1063). Inaddition to isolating QTL alleles in present crop species, thepolynucleotides of the invention are also used to isolate alleles fromthe corresponding QTL of wild relatives.

In addition, the polynucleotides of the present invention can be usedfor marker assisted breeding. Marker assisted breeding uses geneticfingerprinting techniques to assist plant breeders in matching amolecular profile to the physical properties of a variety. This allowsplant breeders to significantly accelerate the speed of natural plantbreeding programs. Marker assisted breeding also allows better retentionof sequences that participate in QTLs.

Following the procedures described above and using a plurality of thepolynucleotides of the present invention, any individual can begenotyped. These individual genotypes are used for the identification ofparticular cultivars, varieties, lines, ecotypes and geneticallymodified plants or can serve as tools for subsequent genetic studiesdirected towards the improvement of multiple phenotypic traits.

4. The Sequences of the Invention

Polynucleotides of the present invention and proteins expressed viatranslation of these polynucleotides are set forth in the SequenceListing, specifically the polynucleotides from Panicum virgatum. TheSequence Listing also provides annotations which are indicative ofuse(s) for which the sequences can be utilized; for example, to maketransgenic plants with modulated growth and phenotype characteristics,including ornamental, biomass and seed yield, confinement, stresstolerance, ornamental and/or compositional characteristics.

5. Use of the Sequences to Make Transgenic Plants

To use the sequences of the present invention or a combination of themor parts and/or mutants and/or fusions and/or variants of them,recombinant DNA constructs are prepared that comprise the polynucleotidesequences of the invention inserted into a vector and that are suitablefor transformation of plant cells. The construct can be made usingstandard recombinant DNA techniques (see, Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1989) New York) and can be introduced into the plantspecies of interest by, for example, Agrobacterium-mediatedtransformation, or by other means of transformation, for example, asdisclosed below.

The vector backbone may be any of those typically used in the field suchas plasmids, viruses, artificial chromosomes, BACs, YACs, PACs andvectors such as, for instance, bacteria-yeast shuttle vectors, lamdaphage vectors, T-DNA fusion vectors and plasmid vectors (see, 17-24).

Typically, the construct comprises a vector containing a nucleic acidmolecule of the present invention with any desired transcriptionaland/or translational regulatory sequences such as, for example,promoters, UTRs, and 3′ end termination sequences. Vectors may alsoinclude, for example, origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, and introns. The vectormay also comprise a marker gene that confers a selectable phenotype onplant cells. The marker may preferably encode a biocide resistancetrait, particularly antibiotic resistance, such as resistance to, forexample, kanamycin, bleomycin, or hygromycin, or herbicide resistance,such as resistance to, for example, glyphosate, chlorsulfuron orphosphinothricin.

It will be understood that more than one regulatory region may bepresent in a recombinant polynucleotide, e.g., introns, enhancers,upstream activation regions, transcription terminators, and inducibleelements. Thus, more than one regulatory region can be operably linkedto said sequence.

To “operably link” a promoter sequence to a sequence, the translationinitiation site of the translational reading frame of said sequence istypically positioned between one and about fifty nucleotides downstreamof the promoter. A promoter can, however, be positioned as much as about5,000 nucleotides upstream of the translation initiation site, or about2,000 nucleotides upstream of the transcription start site. A promotertypically comprises at least a core (basal) promoter. A promoter alsomay include at least one control element, such as an enhancer sequence,an upstream element or an upstream activation region (UAR). For example,a suitable enhancer is a cis-regulatory element (−212 to −154) from theupstream region of the octopine synthase (ocs) gene. Fromm et al. (1989)Plant Cell 1:977-984.

A basal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element that may be locatedbetween about 15 and about 35 nucleotides upstream from the site oftranscription initiation. Basal promoters also may include a “CCAAT box”element (typically the sequence CCAAT) and/or a GGGCG sequence, whichcan be located between about 40 and about 200 nucleotides, typicallyabout 60 to about 120 nucleotides, upstream from the transcription startsite.

The choice of promoters to be included depends upon several factors,including, but not limited to, efficiency, selectability, inducibility,desired expression level, and cell- or tissue-preferential expression.It is a routine matter for one of skill in the art to modulate theexpression of a sequence by appropriately selecting and positioningpromoters and other regulatory regions relative to said sequence.

Some suitable promoters initiate transcription only, or predominantly,in certain cell types. For example, a promoter that is activepredominantly in a reproductive tissue (e.g., fruit, ovule, pollen,pistils, female gametophyte, egg cell, central cell, nucellus,suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo,zygote, endosperm, integument, or seed coat) can be used. Thus, as usedherein a cell type- or tissue-preferential promoter is one that drivesexpression preferentially in the target tissue, but may also lead tosome expression in other cell types or tissues as well. Methods foridentifying and characterizing promoter regions in plant genomic DNAinclude, for example, those described in the following references:Jordano, et al. (1989) Plant Cell 1:855-866; Bustos et al. (1989) PlantCell 1:839-854; Green et al. (1988) EMBO J. 7: 4035-4044; Meier et al.(1991) Plant Cell 3: 309-316; and Zhang et al. (1996) Plant Physiology110: 1069-1079.

Examples of various classes of regulatory regions are described below.Some properties of the regulatory regions indicated below, as well asadditional regulatory regions, are described in more detail in U.S.Patent Application Ser. Nos. 60/505,689; 60/518,075; 60/544,771;60/558,869; 60/583,691; 60/619,181; 60/637,140; 60/757,544; 60/776,307;10/957,569; 11/058,689; 11/172,703; 11/208,308; 11/274,890; 60/583,609;60/612,891; 11/097,589; 11/233,726; 11/408,791; 11/414,142; 10/950,321;11/360,017; PCT/US05/011105; PCT/US05/034308; and PCT/US05/23639.Specifically, the sequences of regulatory regions p326, YP0144, YP0190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633YP0128, YP0275, PT0660, PT0683, PT0758, P70613, PT0672, P70837, YP0092,P0676, PT0708, YP0396, YP0007, YP0111, YP0103, YP0028, YP0121, YP0008,YP0039, YP0115, YP019, YP0120, YP0374, YP0039, YP01, YP0102, YP0110,YP0117, YP0119, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143,YP0156, PT0650, PT0695, P0723, P0838, PT0879, PT0740, PT0535, PT0668,PT0886, PT0585, YP0381, YP0337, YP0374, PT0710, YP0356, YP0385, YP0396,YP0384, PT0688, YP0286, YP0377, PD1367, PT0863, PT0829, PT0665, PT0678,YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, and YP0096 areset forth in the sequence listing of PCT/US06/040572; the sequence ofregulatory region PT0625 is set forth in the sequence listing ofPCT/US05/034343; the sequences of regulatory regions P70623, YP0388,YP0087, YP0093, YP0108, YP0022, and YP0080 are set forth in the sequencelisting of U.S. patent application Ser. No. 11/172,703; the sequence ofregulatory region PR0924 is set forth in the sequence listing ofPCT/US07/62762; and the sequences of regulatory regions p530c10,pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285 are set forth in the sequencelisting of PCT/US06/038236. It will be appreciated that a regulatoryregion may meet criteria for one classification based on its activity inone plant species, and yet meet criteria for a different classificationbased on its activity in another plant species.

Broadly Expressing Promoters: A promoter can be said to be “broadlyexpressing” when it promotes transcription in many, but not necessarilyall, plant tissues. For example, a broadly expressing promoter canpromote transcription of an operably linked sequence in one or more ofthe shoot, shoot tip (apex), and leaves, but weakly or not at all intissues such as roots or stems. As another example, a broadly expressingpromoter can promote transcription of an operably linked sequence in oneor more of the stem, shoot, shoot tip (apex), and leaves, but canpromote transcription weakly or not at all in tissues such asreproductive tissues of flowers and developing seeds. Non-limitingexamples of broadly expressing promoters that can be included in thenucleic acid constructs provided herein include the p326, YP0144,YP0190, p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848,and PT0633 promoters. Additional examples include the cauliflower mosaicvirus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the 1′or 2′ promoters derived from T-DNA of Agrobacterium tumefaciens, thefigwort mosaic virus 34S promoter, actin promoters such as the riceactin promoter, and ubiquitin promoters such as the maize ubiquitin-1promoter. In some cases, the CaMV 35S promoter is excluded from thecategory of broadly expressing promoters.

Root Promoters: Root-active promoters confer transcription in roottissue, e.g., root endodermis, root epidermis, or root vascular tissues.In some embodiments, root-active promoters are root-preferentialpromoters, i.e., confer transcription only or predominantly in roottissue. Root-preferential promoters include the YP0128, YP0275, PT0625,PT0660, PT0683, and PT0758 promoters. Other root-preferential promotersinclude the PT0613, PT0672, PT0688, and PT0837 promoters, which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV 35S promoter (Lam et al., Proc.Natl. Acad. Sci. USA, 86:7890-7894 (1989)), root cell specific promotersreported by Conkling et al., Plant Physiol., 93:1203-1211 (1990), andthe tobacco RD2 promoter.

Maturing Endosperm Promoters: In some embodiments, promoters that drivetranscription in maturing endosperm can be useful. Transcription from amaturing endosperm promoter typically begins after fertilization andoccurs primarily in endosperm tissue during seed development and istypically highest during the cellularization phase. Most suitable arepromoters that are active predominantly in maturing endosperm, althoughpromoters that are also active in other tissues can sometimes be used.Non-limiting examples of maturing endosperm promoters that can beincluded in the nucleic acid constructs provided herein include thenapin promoter, the Arcelin-5 promoter, the phaseolin promoter (Bustoset al., Plant Cell, 1(9):839-853 (1989)), the soybean trypsin inhibitorpromoter (Riggs et al., Plant Cell, 1(6):609-621 (1989)), the ACPpromoter (Baerson et al., Plant Mol. Biol., 22(2):255-267 (1993)), thestearoyl-ACP desaturase promoter (Slocombe et al., Plant Physiol.,104(4):167-176 (1994)), the soybean α′ subunit of β-conglycinin promoter(Chen et al., Proc. Natl. Acad. Sci. USA, 83:8560-8564 (1986)), theoleosin promoter (Hong et al., Plant Mol. Biol., 34(3):549-555 (1997)),and zein promoters, such as the 15 kD zein promoter, the 16 kD zeinpromoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zeinpromoter. Also suitable are the Osgt-1 promoter from the rice glutelin-1gene (Zheng et al., Mol. Cell Biol., 13:5829-5842 (1993)), thebeta-amylase promoter, and the barley hordein promoter. Other maturingendosperm promoters include the YP0092, PT0676, and PT0708 promoters.

Ovary Tissue Promoters: Promoters that are active in ovary tissues suchas the ovule wall and mesocarp can also be useful, e.g., apolygalacturonidase promoter, the banana TRX promoter, the melon actinpromoter, YP0396, and PT0623. Examples of promoters that are activeprimarily in ovules include YP0007, YP0111, YP0092, YP0103, YP0028,YP0121, YP0008, YP0039, YP0115, YP019, YP0120, and YP0374.

Embryo Sac/Early Endosperm Promoters: To achieve expression in embryosac/early endosperm, regulatory regions can be used that are active inpolar nuclei and/or the central cell, or in precursors to polar nuclei,but not in egg cells or precursors to egg cells. Most suitable arepromoters that drive expression only or predominantly in polar nuclei orprecursors thereto and/or the central cell. A pattern of transcriptionthat extends from polar nuclei into early endosperm development can alsobe found with embryo sac/early endosperm-preferential promoters,although transcription typically decreases significantly in laterendosperm development during and after the cellularization phase.Expression in the zygote or developing embryo typically is not presentwith embryo sac/early endosperm promoters. Promoters that may besuitable include those derived from the following genes: Arabidopsisviviparous-1 (see, GenBank No. U93215); Arabidopsis atmycl (see, Urao(1996) Plant Mol. Biol., 32:571-57; Conceicao (1994) Plant, 5:493-505);Arabidopsis FIE (GenBank No. AF129516); Arabidopsis MEA; ArabidopsisFIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No. 6,906,244).Other promoters that may be suitable include those derived from thefollowing genes: maize MACI (see, Sheridan (1996) Genetics,142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) PlantMol. Biol., 22:10131-1038). Other promoters include the followingArabidopsis promoters: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119,YP0137, DME, YP0285, and YP0212. Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

Embryo Promoters: Regulatory regions that preferentially drivetranscription in zygotic cells following fertilization can provideembryo-preferential expression. Most suitable are promoters thatpreferentially drive transcription in early stage embryos prior to theheart stage, but expression in late stage and maturing embryos is alsosuitable. Embryo-preferential promoters include the barley lipidtransfer protein (Ltp1) promoter (Plant Cell Rep (2001) 20:647-654),YP0097, YP0107, YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838,PT0879, and PT0740.

Photosynthetic Tissue Promoters: Promoters active in photosynthetictissue confer transcription in green tissues such as leaves and stems.Most suitable are promoters that drive expression only or predominantlyin such tissues. Examples of such promoters include theribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcSpromoter from eastern larch (Larix laricina), the pine cab6 promoter(Yamamoto et al., Plant Cell Physiol., 35:773-778 (1994)), the Cab-1promoter from wheat (Fejes et al., Plant Mol. Biol., 15:921-932 (1990)),the CAB-1 promoter from spinach (Lubberstedt et al., Plant Physiol.,104:997-1006 (1994)), the cabl R promoter from rice (Luan et al., PlantCell, 4:971-981 (1992)), the pyruvate orthophosphate dikinase (PPDK)promoter from corn (Matsuoka et al., Proc. Natl. Acad. Sci. USA,90:9586-9590 (1993)), the tobacco Lhcb1*2 promoter (Cerdan et al., PlantMol. Biol., 33:245-255 (1997)), the Arabidopsis thaliana SUC2 sucrose-H+symporter promoter (Truernit et al., Planta, 196:564-570 (1995)), andthylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC,FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissue promotersinclude PT0535, PT0668, PT0886, YP0144, YP0380 and PT0585.

Vascular Tissue Promoters: Examples of promoters that have high orpreferential activity in vascular bundles include YP0087, YP0093,YP0108, YP0022, and YP0080. Other vascular tissue-preferential promotersinclude the glycine-rich cell wall protein GRP 1.8 promoter (Keller andBaumgartner, Plant Cell, 3(10):1051-1061 (1991)), the Commelina yellowmottle virus (CoYMV) promoter (Medberry et al., Plant Cell, 4(2):185-192(1992)), and the rice tungro bacilliform virus (RTBV) promoter (Dai etal., Proc. Natl. Acad. Sci. USA, 101(2):687-692 (2004)).

Inducible Promoters: Inducible promoters confer transcription inresponse to external stimuli such as chemical agents or environmentalstimuli. For example, inducible promoters can confer transcription inresponse to hormones such as giberellic acid or ethylene, or in responseto light or drought. Examples of drought-inducible promoters includeYP0380, PT0848, YP0381, YP0337, P0633, YP0374, PT0710, YP0356, YP0385,YP0396, YP0388, YP0384, PT0688, YP0286, YP0377, and PD1367. Examples ofnitrogen-inducible promoters include PT0863, PT0829, PT0665, and PT0886.Examples of shade-inducible promoters include PR0924 and PT0678. Anexample of a promoter induced by salt is rd29A (Kasuga et al. (1999)Nature Biotech 17: 287-291).

Basal Promoters: A basal promoter is the minimal sequence necessary forassembly of a transcription complex required for transcriptioninitiation. Basal promoters frequently include a “TATA box” element thatmay be located between about 15 and about 35 nucleotides upstream fromthe site of transcription initiation. Basal promoters also may include a“CCAAT box” element (typically the sequence CCAAT) and/or a GGGCGsequence, which can be located between about 40 and about 200nucleotides, typically about 60 to about 120 nucleotides, upstream fromthe transcription start site.

Other Promoters: Other classes of promoters include, but are not limitedto, shoot-preferential, callus-preferential, trichome cell-preferential,guard cell-preferential such as PT0678, tuber-preferential, parenchymacell-preferential, and senescence-preferential promoters. Promotersdesignated YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119, andYP0096, as described in the above-referenced patent applications, mayalso be useful.

Other Regulatory Regions: A 5′ untranslated region (UTR) can be includedin nucleic acid constructs described herein. A 5′ UTR is transcribed,but is not translated, and lies between the start site of the transcriptand the translation initiation codon and may include the +1 nucleotide.A 3′ UTR can be positioned between the translation termination codon andthe end of the transcript. UTRs can have particular functions such asincreasing mRNA stability or attenuating translation. Examples of 3′UTRs include, but are not limited to, polyadenylation signals andtranscription termination sequences, e.g., a nopaline synthasetermination sequence. It will be understood that more than oneregulatory region may be present in a recombinant polynucleotide, e.g.,introns, enhancers, upstream activation regions, transcriptionterminators, and inducible elements.

Alternatively, misexpression can be accomplished using a two componentsystem, whereby the first component consists of a transgenic plantcomprising a transcriptional activator operatively linked to a promoterand the second component consists of a transgenic plant that comprise anucleic acid molecule of the invention operatively linked to thetarget-binding sequence/region of the transcriptional activator. The twotransgenic plants are crossed and the nucleic acid molecule of theinvention is expressed in the progeny of the plant (US patentpublication 20050257293A1). In another alternative embodiment of thepresent invention, the misexpression can be accomplished by having thesequences of the two component system transformed in one transgenicplant line.

Another alternative consists in inhibiting expression of a growth orphenotype-modulating polypeptide in a plant species of interest. Theterm “expression” refers to the process of converting geneticinformation encoded in a polynucleotide into RNA through transcriptionof the polynucleotide (i.e., via the enzymatic action of an RNApolymerase), and into protein, through translation of mRNA.“Up-regulation” or “activation” refers to regulation that increases theproduction of expression products relative to basal or native states,while “down-regulation” or “repression” refers to regulation thatdecreases production relative to basal or native states.

A number of nucleic-acid based methods, including anti-sense RNA,ribozyme directed RNA cleavage, and interfering RNA (RNAi) can be usedto inhibit protein expression in plants. Antisense technology is onewell-known method. In this method, a nucleic acid segment from theendogenous gene is cloned and operably linked to a promoter so that theantisense strand of RNA is transcribed. The recombinant vector is thentransformed into plants, as described above, and the antisense strand ofRNA is produced. The nucleic acid segment need not complement the entiresequence of the endogenous gene to be repressed, but typically will besubstantially identical to at least a portion of the endogenous gene tobe repressed. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Typically, a sequence of at least 30nucleotides is used (e.g., at least 40, 50, 80, 100, 200, 500nucleotides or more).

Thus, for example, an isolated nucleic acid provided herein can be anantisense nucleic acid to one of the aforementioned nucleic acidsencoding a growth or phenotype-modulating polypeptide. A nucleic acidthat decreases the level of a transcription or translation product of agene encoding a growth or phenotype-modulating polypeptide istranscribed into an antisense nucleic acid, or complementary to thecoding sequence of the growth or phenotype-modulating polypeptide in theSequence Listing. Alternatively, the transcription product of anisolated nucleic acid can be similar or complementary to the codingsequence of a growth or phenotype-modulating polypeptide in the SequenceListing, but is an RNA that is unpolyadenylated, lacks a 5′ capstructure, or contains an unsplicable intron.

In another method, a nucleic acid can be transcribed into a ribozyme, orcatalytic RNA, that affects expression of an mRNA. (See, U.S. Pat. No.6,423,885). Ribozymes can be designed to specifically pair withvirtually any target RNA and cleave the phosphodiester backbone at aspecific location, thereby functionally inactivating the target RNA.Heterologous nucleic acids can encode ribozymes designed to cleaveparticular mRNA transcripts, thus preventing expression of apolypeptide. Hammerhead ribozymes are useful for destroying particularmRNAs, although various ribozymes that cleave mRNA at site-specificrecognition sequences can be used. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target RNAcontain a 5′-UG-3′ nucleotide sequence. The construction and productionof hammerhead ribozymes is known in the art. See, for example, U.S. Pat.No. 5,254,678 and WO 02/46449 and references cited therein. Hammerheadribozyme sequences can be embedded in a stable RNA such as a transferRNA (tRNA) to increase cleavage efficiency in vivo. Perriman, et al.(1995) Proc. Natl. Acad. Sci. USA, 92(13):6175-6179; de Feyter andGaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, “ExpressingRibozymes in Plants”, Edited by Turner, P. C, Humana Press Inc., Totowa,N.J. RNA endoribonucleases such as the one that occurs naturally inTetrahymena thermophila, and which have been described extensively byCech and collaborators can be useful. See, for example, U.S. Pat. No.4,987,071.

Methods based on RNA interference (RNAi) can be used. RNA interferenceis a cellular mechanism to regulate the expression of genes and thereplication of viruses. This mechanism is thought to be mediated bydouble-stranded small interfering RNA molecules. A cell responds to sucha double-stranded RNA by destroying endogenous mRNA having the samesequence as the double-stranded RNA. Methods for designing and preparinginterfering RNAs are known to those of skill in the art; see, e.g., WO99/32619 and WO 01/75164. For example, a construct can be prepared thatincludes a sequence that is transcribed into an interfering RNA. Such anRNA can be one that can anneal to itself, e.g., a double stranded RNAhaving a stem-loop structure. One strand of the stem portion of a doublestranded RNA comprises a sequence that is similar or identical to thesense coding sequence of the polypeptide of interest, and that is fromabout 10 nucleotides to about 2,500 nucleotides in length. The length ofthe sequence that is similar or identical to the sense coding sequencecan be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25nucleotides to 100 nucleotides. The other strand of the stem portion ofa double stranded RNA comprises an antisense sequence of the growth orphenotype-modulating polypeptide of interest, and can have a length thatis shorter, the same as, or longer than the corresponding length of thesense sequence. The loop portion of a double stranded RNA can be from 10nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000nucleotides, from 20 nucleotides to 500 nucleotides, or from 25nucleotides to 200 nucleotides. The loop portion of the RNA can includean intron. See, e.g., WO 99/53050.

In some nucleic-acid based methods for inhibition of gene expression inplants, a suitable nucleic acid can be a nucleic acid analog. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered morpholino ring, or peptidenucleic acids, in which the deoxyphosphate backbone is replaced by apseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev.,7:187-195; Hyrup et al. (1996) Bioorgan. Med. Chem., 4: 5-23. Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

In some cases, expression of a polypeptide of the invention inhibits oneor more functions of an endogenous polypeptide. For example, a nucleicacid that encodes a dominant negative polypeptide can be used to inhibitgene function. A dominant negative polypeptide typically is mutated ortruncated relative to an endogenous wild type polypeptide, and itspresence in a cell inhibits one or more functions of the wild typepolypeptide in that cell, i.e., the dominant negative polypeptide isgenetically dominant and confers a loss of function. The mechanism bywhich a dominant negative polypeptide confers such a phenotype can varybut often involves a protein-protein interaction or a protein-DNAinteraction. For example, a dominant negative polypeptide can be anenzyme that is truncated relative to a native wild type enzyme, suchthat the truncated polypeptide retains domains involved in binding afirst protein but lacks domains involved in binding a second protein.The truncated polypeptide is thus unable to properly modulate theactivity of the second protein. See, e.g., US 2007/0056058. As anotherexample, a point mutation that results in a non-conservative amino acidsubstitution in a catalytic domain can result in a dominant negativepolypeptide. See, e.g., US 2005/032221. As another example, a dominantnegative polypeptide can be a transcription factor that is truncatedrelative to a native wild type transcription factor, such that thetruncated polypeptide retains the DNA binding domain(s) but lacks theactivation domain(s). Such a truncated polypeptide can inhibit the wildtype transcription factor from binding DNA, thereby inhibitingtranscription activation

Transformation

Nucleic acid molecules of the present invention may be introduced intothe genome or the cell of the appropriate host plant by a variety oftechniques. These techniques, able to transform a wide variety of higherplant species, are well known and described in the technical andscientific literature (see, e.g., 28-29).

A variety of techniques known in the art are available for theintroduction of DNA into a plant host cell. These techniques includetransformation of plant cells by injection (30), microinjection (31),electroporation of DNA (32), PEG (33), use of biolistics (34), fusion ofcells or protoplasts (35), and via T-DNA using Agrobacterium tumefaciens(36-37) or Agrobacterium rhizogenes (38) or other bacterial hosts (39),for example.

In addition, a number of non-stable transformation methods that are wellknown to those skilled in the art may be desirable for the presentinvention. Such methods include, but are not limited to, transientexpression (40) and viral transfection (41).

Seeds are obtained from the transformed plants and used for testingstability and inheritance. Generally, two or more generations arecultivated to ensure that the phenotypic feature is stably maintainedand transmitted.

A person of ordinary skill in the art recognizes that after theexpression cassette is stably incorporated in transgenic plants andconfirmed to be operable, it can be introduced into other plants bysexual crossing. Any of a number of standard breeding techniques can beused, depending upon the species to be crossed.

The methods according to the present invention can be applied to anyplant, preferably higher plants, pertaining to the classes ofAngiospermae and Gymnospermae. Plants of the subclasses of theDicotylodenae and the Monocotyledonae are particularly suitable.Suitable species may come from the family Acanthaceae, Alliaceae,Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae,Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae,Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae,Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae,Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae,Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae,Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae,Solanaceae, Taxaceae, Theaceae, and Vitaceae.

Suitable species may include members of the genus Abelmoschus, Abies,Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

The methods of the present invention are preferably used in plants thatare important or interesting for agriculture, horticulture, biomass forthe production of liquid fuel molecules and other chemicals, and/orforestry. Non-limiting examples include, for instance, Panicum virgatum(switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthusgiganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera(poplar), Zea mays (corn), Glycine max (soybean), Brassica napus(canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryzasativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa),Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet), Panicumspp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp.,Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum(elephant grass), Phalaris arundinacea (reed canarygrass), Cynodondactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartinapectinata (prairie cord-grass), Arundo donax (giant reed), Secalecereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus),Triticosecale spp. (triticum-wheat X rye), Bamboo, Carthamus tinctorius(safflower), Jatropha curcas (jatropha), Ricinus communis (castor),Elaeis guineensis (palm), Linum usitatissimum (flax), Brassica juncea,Manihot esculenta (cassava), Lycopersicon esculentum (tomato), Lactucasativa (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanummelongena (eggplant), Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissativa, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Colchicum autumnale, Veratrum californica,Digitalis lanata, Digitalis pupurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (=Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium,Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata(mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosaspp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia),Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco),Lupinus albus (lupin), Uniola paniculata (oats), bentgrass (Agrostisspp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir),Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (bluegrass),Lolium spp. (ryegrass), Phleum pratense (timothy), and conifers. Ofinterest are plants grown for energy production, so called energy crops,such as cellulose-based energy crops like Panicum virgatum(switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthusgiganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera(poplar), Andropogon gerardii (big bluestem), Pennisetum purpureum(elephant grass), Phalaris arundinacea (reed canarygrass), Cynodondactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartinapectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax(giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.(eucalyptus), Triticosecale spp. (triticum-wheat X rye), and Bamboo; andstarch-based energy crops like Zea mays (corn) and Manihot esculenta(cassava); and sucrose-based energy crops like Saccharum sp. (sugarcane)and Beta vulgaris (sugarbeet); and biodiesel-producing energy crops likeGlycine max (soybean), Brassica napus (canola), Helianthus annuus(sunflower), Carthamus tinctorius (safflower), Jatropha curcas(jatropha), Ricinus communis (castor), Elaeis guineensis (palm), Linumusitatissimum (flax), and Brassica juncea. Thus, the described materialsand methods are useful for modifying biomass characteristics, such ascharacteristics of biomass renewable energy source plants. A biomassrenewable energy source plant is a plant having or producing material(either raw or processed) that comprises stored solar energy that can beconverted to electrical energy, liquid fuels, and other usefulchemicals. In general terms, such plants comprise dedicated energy cropsas well as agricultural and woody plants. Examples of biomass renewableenergy source plants include: Panicum virgatum (switchgrass), Sorghumbicolor (sorghum, sudangrass), Miscanthus giganteus (miscanthus),Saccharum sp. (energycane), Populus balsamifera (poplar), Andropogongerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalarisarundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festucaarundinacea (tall fescue), Spartina pectinata (prairie cord-grass),Medicago sativa (alfalfa), Arundo donax (giant reed), Secale cereale(rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecalespp. (triticum-wheat X rye), Bamboo, Zea mays (corn), Manihot esculenta(cassava), Saccharum sp. (sugarcane), Beta vulgaris (sugarbeet), Glycinemax (soybean), Brassica napus (canola), Helianthus annuus (sunflower),Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinuscommunis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax),and Brassica juncea.

Homologues Encompassed by the Invention

It is known in the art that one or more amino acids in a sequence can besubstituted with other amino acid(s), the charge and polarity of whichare similar to that of the substituted amino acid, i.e. a conservativeamino acid substitution, resulting in a biologically/functionally silentchange. Conservative substitutes for an amino acid within thepolypeptide sequence can be selected from other members of the class towhich the amino acid belongs. Amino acids can be divided into thefollowing four groups: (1) acidic (negatively charged) amino acids, suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids, such as arginine, histidine, and lysine; (3) neutral polar aminoacids, such as serine, threonine, tyrosine, asparagine, and glutamine;and (4) neutral nonpolar (hydrophobic) amino acids such as glycine,alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, cysteine, and methionine.

Nucleic acid molecules of the present invention can comprise sequencesthat differ from those encoding a protein or fragment thereof selectedfrom the group consisting of the nucleotide sequences in the sequencelisting due to the fact that the different nucleic acid sequence encodesa protein having one or more conservative amino acid changes.

Biologically functional equivalents of the polypeptides, or fragmentsthereof, of the present invention can have about 10 or fewerconservative amino acid changes, more preferably about 7 or fewerconservative amino acid changes, and most preferably about 5 or fewerconservative amino acid changes. In a preferred embodiment of thepresent invention, the polypeptide has between about 5 and about 500conservative changes, more preferably between about 10 and about 300conservative changes, even more preferably between about 25 and about150 conservative changes, and most preferably between about 5 and about25 conservative changes or between 1 and about 5 conservative changes.

Identification of Useful Nucleic Acid Molecules and their CorrespondingNucleotide Sequences

The nucleic acid molecules and nucleotide sequences thereof of thepresent invention were identified as functional homologs of othersequences of established function and utility. The other sequences andtheir established function come from public and proprietary sources. Forexample, in some cases the properties of these sequences wereestablished by use of a variety of screens that are predictive ofnucleotide sequences that provide plants with altered size, vegetativegrowth, organ number, plant architecture, biomass, and/or enhancedresistance to various abiotic or biotic stresses. In other cases,characteristics of similar sequences are known from scientific andtechnical literature readily accessible to those of skill in the art.

Functional homologs/orthologs from Panicum virgatum for each gene ofinterest, which in many cases may be capable of modulating growth andphenotype characteristic when transformed in plants. The “Determinationof Functional Homolog/Ortholog Sequences” process described below can beused to identify functional homologs/orthologs. Functionalhomologs/orthologs of a sequence of interest are understood to affectsimilar phenotype(s) as observed for the respective sequence of interestwhen their expression is modulated in plants.

Determination of Functional Homolog/Ortholog Sequences

A subject sequence is considered a functional homolog or ortholog of aquery sequence if the subject and query sequences encode proteins havinga similar function and/or activity. A process known as Reciprocal BLAST(Rivera et al., Proc. Natl. Acad. Sci. USA, 95:6239-6244 (1998)) wasused, for many of the sequences provided, to identify potentialfunctional homolog and/or ortholog sequences from a database consistingof Ceres-Inc. proprietary peptide sequences from Panicum virgatum.

Before starting a Reciprocal BLAST process, a specific query polypeptideis searched against all peptides from its source species using BLAST inorder to identify polypeptides having BLAST sequence identity of 80% orgreater to the query polypeptide and an alignment length of 85% orgreater along the shorter sequence in the alignment. The querypolypeptide and any of the aforementioned identified polypeptides aredesignated as a cluster.

The BLASTP version 2.0 program from Washington University at SaintLouis, Mo., USA is used to determine BLAST sequence identity andE-value. The BLASTP version 2.0 program includes the followingparameters: 1) an E-value cutoff of 1.0e-5; 2) a word size of 5; and 3)the -postsw option. The BLAST sequence identity is calculated based onthe alignment of the first BLAST HSP (High-scoring Segment Pairs) of theidentified potential functional homolog and/or ortholog sequence with aspecific query polypeptide. The number of identically matched residuesin the BLAST HSP alignment is divided by the HSP length, and thenmultiplied by 100 to get the BLAST sequence identity. The HSP lengthtypically includes gaps in the alignment, but in some cases gaps can beexcluded.

The main Reciprocal BLAST process consists of two rounds of BLASTsearches; forward search and reverse search. In the forward search step,a query polypeptide sequence, “polypeptide A,” from source species SA(such as Arabidopsis) is BLASTed against all Ceres-Inc. proprietarypeptide sequences from Panicum virgatum. Top hits are determined usingan E-value cutoff of 10-5 and a sequence identity cutoff of 35%. Amongthe top hits, the sequence having the lowest E-value is designated asthe best hit, and considered a potential functional homolog or ortholog.Any other top hit that has a high BLAST sequence identity to the besthit or to the original query polypeptide is considered a potentialfunctional homolog or ortholog as well.

In the reverse search round, the top hits identified in the forwardsearch from Panicum virgatum are BLASTed against all protein sequencesfrom the source species SA. A top hit from the forward search thatreturns a polypeptide from the aforementioned cluster as its best hit isalso considered as a potential functional homolog or ortholog.

Functional homologs and/or orthologs are identified by manual inspectionof potential functional homolog and/or ortholog sequences. In some casesidentification is based on known correlations between sequence domainsand functions for specific classes of biomolecules.

Information in the Sequence Listing

The switchgrass sequences provided in the Sequence Listing are annotatedto indicate one or several potential applications of the respectivesequences. Some sequences are enzymes, i.e. catalysts of specificchemical or biochemical reactions, and their activity is indicated byenzyme classification (EC) numbers. The EC numbers used in the sequencelisting correspond to the swissprot enzyme classification system, asfound on the World Wide Web. Some sequences contain “pfam” domains whichare indicative of particular applications. The specific pfam domains aredescribed in more detail by various sources on the World Wide Web.

Some switchgrass sequences in the Sequence Listing are annotated in the“miscellaneous features” section as functional homologs of knownsequences, and associated traits that could be modulated by therespective sequences in transgenic plants. In this context “great-hit”denotes a BLAST hit from a forward search that returns a polypeptidefrom the query cluster as its best hit in the reverse BLAST search, asexplained above. Known sequence-function associations are sometimesbased at least in part on literature documentation. In some cases, theknown query sequences are not reproduced in the sequence listing, but itis identified by literature reference, such as by reference to specificsequences in patent publications or Arabidopsis locus numbers. Ifdesired, sequence information for these designations may be obtainedfrom various sources, such as the EMBL sequence database or Arabidopsisdatabase available on the World Wide Web. In some cases, query sequencessuch as SEQ ID NOs: 9177-9762 are provided in the sequence listing,along with information pertinent to observed transgenic plant traits.When a listed query sequence is a truncation of a known coding region,those of skill in the art could design if necessary an equivalenttruncation of the corresponding switchgrass functional homologs toobtain the indicated phenotype.

Modulated growth and phenotype characteristics for some of the sequencesof the invention are noted by entries in the “miscellaneous feature”section for the respective nucleic acid and/or polypeptide sequence inthe Sequence Listing. Valuable applications of the respective sequencesare also sometimes noted in the Sequence Listing. In many cases, traitswere originally associated with known sequences by misexpression orother transgenic or genetic interference with specific gene functions.For some sequences, plants were transformed with the genes of interestand screened for modulated morphological characteristics, as described,for example in PCT/US2005023326 or PCT/US2005/047099. When applicable,phenotypic observations for transformants are noted in the SequenceListing. For some sequences, transgenic trait associations weresuggested by results of screening transgenic plants for tolerance tospecific stress conditions. Screening procedures and some results areexemplified in PCT/US2005/018950, PCT/US2005/018912, PCT/US2005/014197,or PCT/US2006/040572. Other methods are exemplified by references citedin the Sequence Listing or below.

From the disclosure of the Sequence Listing, it can be seen that thenucleotides and polypeptides of the inventions are useful, dependingupon the respective individual sequence, to make plants with one or morealtered characteristics in traits. For example, the sequences can beused, as noted in the sequence listing for the respective sequences, forbiocontainment applications, for making ornamental plants with delayedpetal abscission, alkaloid content, amino acid content, carbohydratecontent, carbon content, carotene content, cell wall content and/orcomposition, glycerol content, lignin content, lutein content, lycopenecontent, nitrogen content, oil content, protein content, sterol content,terpenoid content, tocopherol content, enhanced nutritional value, highaluminum sensitivity, abiotic stress sensitivity, abscission, asexualembryo production, biomass, biotic stress sensitivity, carbohydratecontent, carbon-nitrogen partitioning, cold sensitivity, droughtsensitivity, endosperm cell number, endosperm cell size, endosperm size,flowering time, growth rate, heat sensitivity, herbicide sensitivity,high pH sensitivity, light quality response, light quality sensitivity,light response, light sensitivity, low iron sensitivity, low lightsensitivity, nitrogen use efficiency, oxidative stress sensitivity,phosphate use efficiency, plant growth and development, rootdevelopment, salt sensitivity, seed content, seed number, seed quality,seed size, seedling growth, silicon content, time to senescence, UVsensitivity, water use efficiency, yield.

Additionally, clone ids 1986288 (SEQ ID NO: 8028) and 1792286 (SEQ IDNO: 3570), are “useful for making plants with modulated flowering time.”Furthermore, clone ids 1769278 (SEQ ID NO: 1674), 1797688 (SEQ ID NO:4222), and 1891486 (SEQ ID NO: 6912) are “useful for making plants withmodulated drought sensitivity.”

Nucleotides and polypeptides that are useful for modulating plantcharacteristics in traits such as sterility, lethality and/or viabilityhave been noted as “useful for biocontainment applications” or “usefulfor making plants with modulated asexual embryo production” in theSequence Listing. Nucleotides and polypeptides that with thisdesignation include those that are able to confer one or more of thefollowing phenotypes, relative to wild-type control, when mis-expressedin plants: increased or decreased number of floral organs; alter floralorgan type; reduced fertility; sterility, including female-sterilityand/or male-sterility; alter how leaves emerge from the meristem; low/noseed germination; and reduced plant viability (e.g. albino plants andplants with vitrified leaves). The ability to modulate sterility,lethality, and/or viability is important in developing a geneticconfinement system designed to reduce or prevent gene flow fromtransgenic pants to commercial crops and wild-type counterparts, makingornamental plants; and for other agricultural and/or horticulturalpurposes. Nucleotides and polynucleotides useful for developing agenetic confinement system can be utilized by procedures known to thoseskilled in the art, such as in US2005/0257293 A1, hereby incorporated byreference.

Some nucleotides and polypeptides are noted as being “useful for makingplants with modulated biomass”, “useful for making plants with modulatedyield”, “useful for making plants with modulated flowering time,”“useful for making plants with modulated seed size”, “useful for makingplants with modulated time to senescence”, “useful for making plantswith modulated endosperm cell number”, “useful for making plants withmodulated endosperm cell size”, “useful for making plants with modulatedendosperm size”, “useful for making plants with modulated growth rate”,“useful for making plants with modulated seed content”, “useful formaking plants with modulated seed number”, “useful for making plantswith modulated seed quality”, “useful for making plants with modulatedplant growth and development.” or “useful for making plants withmodulated seedling growth” in the Sequence Listing. Nucleotides andpolypeptides that have been given these designation include those thatare able to confer one or more of the following phenotypes, relative towild-type control, when mis-expressed in plants: increased or decreasedplant size; increased or decreased plant height; increased or decreasedleaf size; altered leaf shape; altered leaf structure; increased ordecreased number of leaves; increased or decreased organ size; alteredorgan shape; increased or decreased organ number, increased or decreasedbranching length; increased or decreased branch number, increase ordecrease petal abscission, increase or decrease in leaf abscission,increased or decreased apical dominance; and increased or decreasedhypocotyls length. Altering plant biomass is valuable for increasingplant biomass produced per acre of arable land, increasing crop yieldper acre of arable land, utilizing plants as chemical factories toproduce valuable pharmaceutical compounds, developing a geneticconfinement system designed to reduce or prevent gene flow fromtransgenic plants to commercial crops and wild-type counterparts, makingornamental plants; and for other agricultural and/or horticulturalpurposes.

Some nucleotides and polypeptides that are useful for modulating plantcharacteristics in traits such as the composition of a plant, plantmaterial, plant tissue, plant cell and seed from a plant include thosethat have been given the designations “useful for making plants withaltered alkaloid content”, “useful for making plants with altered carboncontent”, “useful for making plants with modulated carbon-nitrogenpartitioning”, “useful for making plants with altered carotene content”,“useful for making plants with modulated carbohydrate content”, “usefulfor making plants with altered carbohydrate content”, “useful for makingplants with altered cell wall content and/or composition”, “useful formaking plants with altered glycerol content”, “useful for making plantswith altered lignin content”, “useful for making plants with alteredlutein content”, “useful for making plants with altered lycopenecontent”, “useful for making plants with altered nitrogen content”,“useful for making plants with altered oil content”, “useful for makingplants with altered protein content”, “useful for making plants withaltered amino acid content”, “useful for making plants with alteredsterol content”, “useful for making plants with altered terpenoidcontent”, “useful for making plants with altered tocopherol content”,“useful for making plants with modulated silicon content”, or “usefulfor making plants with enhanced nutritional value” in the SequenceListing. Nucleotides and polypeptides that have been given thesedesignations include those that are able to confer one or more of thefollowing phenotypes, relative to wild-type control, when mis-expressedin plants: increased or decreased carbon content; increased or decreasedplant nitrogen content; altered color (indicative of change(s) to thechemical composition); altered metabolic profile, increased or decreasedstarch content, increased or decreased fiber content; increased ordecreased amount of a valuable compound (e.g. increased alkaloids and/orterpenoids); increased or decreased number of trichomes; increased ordecreased cotyledon size; increased or decreased cotyledon number;altered cotyledon shape; increased or decreased fruit size; increased ordecreased fruit length; altered fruit shape; increased or decreased seedsize; and altered seed shape; altered seed color (indicative of alteredchemical composition); and having activated expression of a geneoperably linked to an alkaloid or terpenoid related regulatory region orpromoter. Altering characteristics such as the composition of a plant,plant organ, plant tissue and plant cell is valuable for improving thenutritional value of crops, improving the composition of plants to beused as bio-fuels, utilizing plants as chemical factories by increasingthe content of valuable pharmaceutical compounds, producing plants withincreased tolerance to abiotic or biotic stress, developing a geneticconfinement system designed to reduce or prevent gene flow fromtransgenic plants to commercial crops and wild-type counterparts, makingornamental plants, and for other agricultural and/or horticulturalpurposes. Nucleotides and polynucleotides useful for developing plantswith modified compositions can be utilized by procedures known to thoseskilled in the art, such as in PCT/US2006/014161, PCT/US2005/032680,PCT/US2006/0022851, PCT/US2005/44112, PCT/US2005/043562,PCT/US2006/41516, U.S. Ser. No. 60/838,646, U.S. Ser. No. 60/855,108,U.S. Ser. No. 60/854,825 PCT/US2006/360,459, PCT/US2007/061052, andPCT/US2007/002214, hereby incorporated by reference.

Some nucleotides and polypeptides that are useful for modulating plantcharacteristics in traits such as phosphate use include those that havebeen given the designation “Useful for making plants with modulatedphosphate use efficiency”, “useful for making plants with modulated highpH sensitivity”, or “useful for making plants with high aluminumsensitivity”, in the Sequence Listing. Nucleotides and polypeptides thathave been given these designations include those that are able to conferone or more of the following phenotypes, relative to wild-type control,when mis-expressed in plants: increased or decreased tolerance to lowphosphate conditions; increased or decreased tolerance to no phosphateconditions, and increased or decreased tolerance to high pH conditions.Altering characteristics such as phosphate use through genetictechnologies is valuable for producing crop plants with increasedtolerance to phosphate limiting conditions, using traditionallyun-arable land to grow crop plants with increased tolerance to phosphatelimiting conditions, developing a genetic confinement system designed toreduce or prevent gene flow from transgenic plants to commercial cropsand wild-type counterparts, and for other agricultural and/orhorticultural purposes. Nucleotides and polynucleotides useful fordeveloping plants with modulated phosphate use efficiency can beutilized by procedures known to those skilled in the art, such as inPCT/US2005/018912, hereby incorporated by reference.

Nucleotides and polypeptides that are useful for modulating plantcharacteristics in traits such as light responses include those thathave been given the designations “useful for making plants withmodulated light quality response”, “useful for making plants withmodulated light quality sensitivity”, “useful for making plants withmodulated light response”, “useful for making plants with modulatedlight sensitivity”, “useful for making plants with modulated UVsensitivity”, “useful for making plants with modulated low lightsensitivity” in the Sequence Listing. Nucleotides and polypeptides thathave been given these designations include those that are able to conferone or more of the following phenotypes, relative to wild-type control,when mis-expressed in plants: increased or decreased vigor in the dark;increased or decreased seedling vigor under low light conditions;increased or decreased plant vigor under low light conditions; increasedor decreased leaf length; altered leaf shape; altered leaf structure,and increased or decreased cotyledon length. Altering characteristicssuch as shade avoidance and shade tolerance through genetic technologiesis valuable for producing plants with tolerance to light limitingconditions, increasing plant biomass produced per acre of arable land,increasing crop production per acre of arable land, developing a geneticconfinement system designed to reduce or prevent gene flow fromtransgenic plants to commercial crops and wild-type counterparts, makingornamental plants, and for other agricultural and/or horticultural 1 s.Nucleotides and polynucleotides useful for developing plants with lightresponses can be utilized by procedures known to those skilled in theart, such as in U.S. Ser. No. 60/799,404, U.S. Ser. No. 60/813,533, andU.S. Ser. No. 60/818,569, hereby incorporated by reference.

Nucleotides and polypeptides that are useful for modulating plantcharacteristics in traits such as nitrogen use include those that havebeen given the designation “Useful for making plants with modulatednitrogen use efficiency” in the Sequence Listing. Nucleotides andpolypeptides that have been given the “Nitrogen use” designation includethose that are able to confer one or more of the following phenotypes,relative to wild-type control, when mis-expressed in plants: increasedor decreased tolerance to low nitrogen conditions and surrogate lownitrogen conditions (e.g. exposure to an effective amount of MSX); in tor decreased tolerance to no nitrogen conditions; increased tolerance tohigh nitrogen conditions. Altering nitrogen use through genetictechnologies is valuable for producing plants with increased toleranceto high or low nitrogen conditions, decreasing the amount of fertilizersused in crop production, using traditionally un-arable land to grow cropplants with increased tolerance to high or low nitrogen conditions,developing a genetic confinement system designed to reduce or preventgene flow from transgenic plants to commercial crops and wild-typecounterparts, and for other agricultural and/or horticultural purposes.Nucleotides and polynucleotides useful for developing plants withimproved nitrogen use efficiency can be utilized by procedures known tothose skilled in the art, such as in PCT/US2005/014197, herebyincorporated by reference.

Nucleotides and polypeptides that are useful for modulating plantcharacteristics in traits such as stress tolerance include those thathave been given the designations “useful for making plants withmodulated cold sensitivity”, “useful for making plants with modulateddrought sensitivity”, “useful for making plants with modulated heatsensitivity”, “useful for making plants with modulated low ironsensitivity”, “useful for making plants with modulated oxidative stresssensitivity”, “useful for making plants with modulated rootdevelopment”, “useful for making plants with modulated water useefficiency”, “useful for making plants with modulated salt sensitivity”,“useful for making plants with modulated abiotic stress sensitivity”, or“useful for making plants with modulated biotic stress sensitivity” inthe Sequence Listing. Nucleotides and polypeptides that have been giventhese designation include those that are able to confer one or more ofthe following phenotypes, relative to wild-type control, whenmis-expressed in plants: increased or decreased tolerance to droughtand/or surrogate drought conditions (e.g. exposure to effective amountsof ABA, PEG, mannitol or sucrose); increased or decreased tolerance tolow temperature conditions; increased or decreased tolerance to hightemperature conditions; increased or decreased salt tolerance; increasedor decreased tolerance to oxidative stressors and/or su oxidativestressors (e.g. exposure to an effective amount of arginine, ozone, orsalicylic acid); having leaves with shiny or dull appearance (indicativeof altered wax composition and/or content); and increase or decreasepest resistance. Altering abiotic stress tolerance through genetictechnologies is valuable for farmers seeking to minimize economic lossesdue to drought, cold, heat, flooding and oxidative stressors; producingcrop plants with increased tolerance to abiotic stressors; usingtraditionally un-arable land to grow crop plants with increasedtolerance to abiotic stressors; developing a genetic confinement systemdesigned to reduce or prevent gene flow from transgenic plants tocommercial crops and wild-type counterparts; making ornamental plants;and for other agricultural and/or horticultural purposes. Nucleotidesand polynucleotides useful for developing plants with improved abioticstress tolerance can be utilized by procedures known to those skilled inthe art, such as in PCT/US2005/018950, U.S. Ser. No. 11/248,547, U.S.Ser. No. 60/837,434, U.S. Ser. No. 60/860,296, or U.S. Ser. No.60/851,585, hereby incorporated by reference.

The phenotypes mentioned in the sequence listing can be modulated bycontrolling the expression of nucleic acid sequences and polypeptidesequences that confer phenotype(s) when mis-expressed in plants.Modulation of a phenotype can also be achieved by inhibiting theexpression of nucleic acid sequences and polypeptide sequences thatconfer phenotype(s) when mis-expressed in plants. A phenotype resultingfrom the expression of a nucleic acid sequence and/or polypeptidesequence can be modulated (e.g. increase or decrease of anobservable/measurable phenotypic change in relation to wild-typecontrol) using recombinant-DNA methods, as discussed in previousparagraphs.

According to another aspect, the nucleotide sequences of the inventionencode polypeptides that can be utilized as herbicide targets, thoseuseful in the screening of new herbicide compounds. Thus, the proteinsencoded by the nucleotide sequences provide the bases for assaysdesigned to easily and rapidly identify novel herbicides.

According to yet another aspect, the present invention provides a methodof identifying a herbicidal compound, comprising: (a) combining apolypeptide comprising an amino acid sequence at least 85% identical toan amino acid sequence selected from the group consisting of thepolypeptides described in the sequence listing with a compound to betested for the ability to inhibit the activity of said polypeptide,under conditions conducive to inhibition; (b) selecting a compoundidentified in (a) that inhibits the activity of said polypeptide; (c)applying a compound selected in (b) to a plant to test for herbicidalactivity; (d) selecting a compound identified in (c) that has herbicidalactivity. The polypeptide can alternatively comprise an amino acidsequence at least 90%, or at least 95%, or at least 99% identical to anamino acid sequence selected from the group consisting of thepolypeptides in the sequence listing. The present invention alsoprovides a method for killing or inhibiting the growth or viability of aplant, comprising applying to the plant a herbicidal compound identifiedaccording to this method.

The Sequence Listing sets forth the polypeptide and polynucleotidesequences of the invention, including, in many cases, functionalhomologs of specific query sequences. The Sequence Listing indicateswhich of the functional homologs are associated with each querysequence. Sequencing reads from the same clone are given the same cloneid number. The 3′ read of a clone will additionally have the “R”designation. Overlapped clones and 5′ reads of a clone do not have the“R” designation.”

The present invention further encompasses nucleotides that encode theabove described polypeptides, such as those included in the sequencelisting, as well as the complements and/or fragments thereof, andincluding alternatives thereof based upon the degeneracy of the geneticcode.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

REFERENCES

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1. A nucleic acid molecule comprising: (a) a nucleotide sequenceencoding an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO: 76; (b) a nucleotide sequence that isat least 90% identical to the nucleotide sequence of SEQ ID NO: 75; (c)a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 76;(d) a nucleotide sequence comprising the nucleotide sequence of SEQ IDNO:75; and wherein the nucleic acid molecule is operably linked to aheterologous regulatory region.
 2. (canceled)
 3. A vector, comprisingthe nucleic acid molecule of claim
 1. 4. A method of modulating plantgrowth, development or phenotype characteristics, said method comprisingintroducing into a plant cell a nucleic acid molecule according to claim1, wherein a plant produced from said plant cell has modulated plantgrowth, development or phenotype characteristics as compared to thecorresponding level of said characteristic in tissue of a control plantthat does not comprise said nucleic acid molecule.
 5. The method ofclaim 4, wherein the modulated plant growth, development or phenotypecharacteristics comprise a modulation in any one of plant size, plantheight, plant strength, vegetative growth, color, plant architecture,amount of branching, branching angle, branching length, number or leavesper branch, organ number, organ size, organ shape, leaf shape, leafstructure, leaf size, leaf number, leaf angle, biomass, sterility,seedling lethality, seed weight, seed size, seed color, seed yield, seedgermination, seed content, seed structure, seed carbon content, seednitrogen content, seed fiber content, fruit composition, fruit shape,fruit size, fruit length, fruit yield, silique length, silique shape,flower length, flower shape, flower size, inflorescence length,inflorescence thickness, cotyledon size, cotyledon number, cotyledonshape, crop development or harvest time, flowering time, senescence,time to bolting, drought or stress tolerance, biotic stress tolerance,abiotic stress tolerance, tolerance to high density plant population,tolerance to high pH, tolerance to low pH, tolerance to low nitrogenconditions, tolerance to no nitrogen conditions, tolerance to highnitrogen conditions, tolerance to nutrient limiting conditions,tolerance to oxidative stress, tolerance to cold stress, tolerance toheat stress, tolerance to saltstress, chlorophyll content,photosynthetic capacity, root growth, nutrient uptake, chemicalcomposition, anthocyanin content, starch content, nitrogen content,internode length, hypocotyls length, ability to grow in shade, and shadeavoidance as compared to the corresponding characteristics of a controlplant that does not comprise said nucleic acid.
 6. (canceled)
 7. Themethod of claim 1, wherein said regulatory region is a promoter selectedfrom the group consisting of YP0092, PT0676, PT0708, PT0613, PT0678,PT0688, PT0837, the napin promoter, the Arcelin-5 promoter, thephaseolin gene promoter, the soybean trypsin inhibitor promoter, the ACPpromoter, the stearoyl-ACP desaturase gene, the soybean a′ subunit of13-conglycinin promoter, the oleosin promoter, the 15 kD zein promoter,the 16 kD zein promoter, the 19 kD zein promoter, the 22 kD zeinpromoter, the 27 kD zein promoter, the Osgt-1 promoter, the beta-amylasegene promoter, the barley hordein gene promoter, p326, YP0144, YP0190,p13879, YP0050, p32449, 21876, YP0158, YP0214, YP0380, PT0848, PT0633,the cauliflower mosaic virus 35S promoter, the mannopine synthasepromoter, the 1 or 2′ promoters derived from T-DNA of Agrobacteriumtumefaciens, the figwort mosaic virus 34S promoter, actin promoters suchas the rice actin promoter, ubiquitin promoters such as the maizeubiquitin-1 promoter, ribulose-1,5-bisphosphate carboxylase promoterssuch as the RbcS promoter from eastern larch (Larix laricina), the pinecab6 promoter, the Cab-1 gene promoter from wheat, the CAB-1 promoterfrom spinach, the cabl R promoter from rice, the pyruvate orthophosphatedikinase (PPDK) promoter from corn, the tobacco Lhcb1*2 promoter, theArabidopsis thaliana SUC2 sucrose-H+ symporter promoter, and thylakoidmembrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR,atpC, atpD, cab, rbcS, PT0535, PT0668, PT0886, PR0924, and PT05851.
 8. Aplant cell comprising the nucleic acid molecule of claim 1, wherein aplant produced from said plant cell has modulated plant growth,development or phenotype characteristics as compared to a control plantthat does not comprise said nucleic acid molecule.
 9. (canceled)
 10. Atransgenic plant comprising the plant cell of claim
 8. 11. Progeny ofthe plant of claim 10, wherein said progeny comprises said nucleic acidmolecule and has modulated plant growth, development or phenotypecharacteristics as compared to a control plant that does not comprisesaid nucleic acid molecule.
 12. Seed from a transgenic plant accordingto claim 10 wherein said seed comprises said nucleic acid molecule. 13.Vegetative tissue from a transgenic plant according to claim 10, whereinsaid seed comprises said nucleic acid molecule.
 14. A food productcomprising vegetative tissue from a transgenic plant according to claim10.
 15. A feed product comprising vegetative tissue from a transgenicplant according to claim
 10. 16. A method for detecting a nucleic acidin a sample, comprising: (a) providing an nucleic acid moleculeaccording to claim 1; (b) contacting said nucleic acid molecule with asample under conditions that permit a comparison of the nucleotidesequence of the isolated nucleic acid with a nucleotide sequence ofnucleic acid in the sample; and (c) analyzing the comparison.
 17. Amethod for modulating plant growth, development or phenotypecharacteristics in a plant, comprising: (a) transforming a plant with anucleic acid molecule according to claim 1; and (b) expressing saidnucleotide sequence in said transformed plant, whereby said transformedplant has modulated plant growth, development or phenotypecharacteristics as compared to a plant that has not been transformedwith said nucleotide sequence.
 18. A method for modulating the growth,development or phenotype characteristics of a plant, said methodcomprising altering the level of expression in said plant of a nucleicacid molecule according to claim
 1. 19. A transgenic plant havingmodified growth, development or phenotype characteristics as compared toa wild-type plant of the same species, wherein the transgenic plantcomprises a recombinant polynucleotide comprising the nucleic acidmolecule of claim 1, and wherein the polypeptide encoded by saidnucleotide sequence confers to the transgenic plant modified growth,development or phenotype characteristics as compared to a wild-typeplant of the same species.
 20. A method for producing a transgenic planthaving modified growth, development or phenotype characteristics ascompared to a wild-type plant of the same species, the method stepscomprising: (a) inserting the nucleic acid molecule of claim 1 into anexpression vector; (b) introducing the vector into a plant or a cell ofa plant to overexpress the encoded polypeptide of the nucleotidesequence; and (c) selecting the transgenic plant for modified growth,development or phenotype characteristics as compared to the wild-typeplant of the same species. 21-23. (canceled)